Expression of self-incompatibility ribonucleases of Antirrhinum in Escherichia coli

Self-incompatibility is an intraspecific reproductive barrier to prevent self-fertilization inthe flowering plants. In many species, self-incompatibility is controlled by a single S locus with multiple alleles. So far, the only gene known in the S locus of the Solanaceae, Scrophulariaceae and Rosaceae encodes a class of ribonucleases, called self-incompatibility ribonucleases (S RNases), which have been shown to mediate stylar expression of self-incompatible reaction. As the first step to investigate their three-dimensional structure, we successfully expressed three biologically active S RNases of Antirrihnum (S2, S4 and S5) in Escherichia coli (E. coli). Their functional expressions caused no detrimental effect on host bacteria growth and provided a basis for a large scale preparation of S RNase proteins. Possible reasons for non-lethality of S RNases on E. coliare discussed.

Poly(A)-specific ribonuclease protein promotes the proliferation,invasion and migration of esophageal cancer cells

BACKGROUND Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease(PARN)gene in gastric cancer,head and neck squamous cell carcinoma,melanoma,cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis.The expression of the PARN gene in esophageal cancer(EC)tissue is also significantly higher than that in normal tissues,but the effect of PARN on the proliferation,migration and invasion of EC cells remains unclear.AIM To investigate the relationship between PARN and the proliferation,migration and invasion of EC cells.METHODS The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected.PARN mRNA levels were measured using a tissue microarray,and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients.In addition,the effects of PARN gene knockout on tumor cell proliferation,invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1,and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model.RESULTS The expression of PARN in EC tissues was higher than that in adjacent normal tissues,and the level of PARN expression was significantly positively correlated with lymphatic metastasis.Patients with high PARN levels had poor overall survival.BIM,IGFBP-5 and p21 levels were significantly increased in the PARN knockout group,while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data.In addition,the expression levels of Akt,p-Akt,PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased.The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased,the growth and proliferation of tumor cells were significantly inhibited,and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout.In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA(sh-NC)and PARN shRNA(sh-PARN)showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC,indicating that PARN knockdown significantly inhibited tumor growth in vivo.CONCLUSION PARN has antiapoptotic effects on EC cells and promotes their proliferation,invasion and migration,which is associated with the development of EC and poor patient prognosis.PARN may become a potential target for the diagnosis,prognosis prediction and treatment of EC.

粟酒裂殖酵母N-糖酰胺酶在大肠杆菌中的表达、纯化及活性分析

根据GenBank中公布的粟酒裂殖酵母(Schizosaccharomyces pombe)N-糖酰胺酶(Pnglp)cDNA序列,设计并合成一对特异性引物,利用RT-PCR技术从粟酒裂殖酵母中克隆出糖酰胺酶cDNA。将得到的基因克隆到表达载体pET-15b中。重组质粒转入大肠杆菌BL21(DE3)中,经诱导表达和纯化提取后,进行酶活测定。实验结果表明,该酶的分子量约为39 kD,纯化后的重组N-糖酰胺酶可以对变性处理的糖蛋白进行糖链的切除,且这种作用需要还原剂DTT的辅助作用;N-糖酰胺酶只对错误折叠的糖蛋白有作用,对天然的糖蛋白没有作用。等量粟酒裂殖酵母Png1p在不同温度、pH、DTT浓度和底物变性温度下对等量核糖核酸酶B(RNase B)的脱糖基化检测发现,重组酶的最适反应温度30℃,最适反应pH为7.0,需要的最适DTT浓度为10 mmol/L,底物在100℃处理10 min时酶的脱糖基化率最高。

Human ribonuclease 9,a member of ribonuclease A superfamily,specifically expressed in epididymis,is a novel sperm-binding protein

To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences.According to the determined mature protein,recombinant human RNase 9 was prepared in E.coli.Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected,and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay.The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA,but exhibited antibacterial activity,in a concentration/time dependent manner,against E.coli.Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis,but not present in other tissues examined,and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa.These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.

A highly sensitive bio-barcode immunoassay for multi-residue detection of organophosphate pesticides based on fluorescence antiquenching

Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal enables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concentration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.

Role of Pathogen-Related Protein 10 (PR 10) under Abiotic and Biotic Stresses in Plants

Members of the Pathogenesis Related(PR)10 protein family have been identified in a variety of plant species and a wide range of functions ranging from defense to growth and development has been attributed to them.PR10 protein possesses ribonuclease(RNase)activity,interacts with phytohormones,involved in hormone-mediated signalling,afforded protection against various phytopathogenic fungi,bacteria,and viruses particularly in response to biotic and abiotic stresses.The resistance mechanism of PR10 protein may include activation of defense signalling pathways through possible interacting proteins involved in mediating responses to pathogens,degradation of RNA of the invading pathogens.Moreover,several morphological changes have been shown to accompany the enhanced abiotic stress tolerance.In this review,the possible mechanism of action of PR10 protein against biotic and abiotic stress has been discussed.Furthermore,our findings also confirmed that the in vivo Nitric oxide(NO)is essential for most of environmental abiotic stresses and disease resistance against pathogen infection.The proper level of NO may be necessary and beneficial,not only in plant response to the environmental abiotic stress,but also to biotic stress.The updated information on this interesting group of proteins will be useful in future research to develop multiple stress tolerance in plants.

Arg462Gln and Asp541Glu polymorphisms in ribonuclease L and prostate cancer risk:a meta-analysis

Objective：The association between ribonuclease L（RNASEL）gene polymorphisms and prostate cancer risk has been widely reported,but the results of these studies remained controversial and underpowered.We performed a meta-analysis of 28 studies to evaluate the association between Arg462Gln and Asp541Glu polymorphisms in the RNASEL gene and prostate cancer risk.Methods：Odds ratios（ORs）with 95%confidence intervals（CIs） were estimated to assess the association between RNASEL polymorphisms and prostate cancer risk.Results：A significantly increased prostate cancer risk was found for the Arg462Gln polymorphism in Africans（Gln/Gln vs Arg/Arg：OR=2.50,95%CI=1.28-4.87;Gln/Gln vs Gln/Arg＋Arg/Arg：OR=2.54,95%CI=1.30-4.95）,but not in Europeans and Asians.Additionally,the Asp541Glu polymorphism was associated with increased total prostate cancer risk（Glu-allele vs Asp-allele：OR=1.04,95%CI=1.01-1.07;Glu/Glu vs Asp/Asp：OR=1.22,95%CI= 1.03-1.46;Glu/Glu vs Glu/Asp＋Asp/Asp：OR=1.09,95%CI=1.02-1.16）.In the stratified analysis for the Asp541Glu polymorphism,there was a significantly increased prostate cancer risk in Africans and Europeans,and in hospital-based prostate cancer cases.Conclusion：The meta-analysis results showed evidence that RNASEL Arg462Gln and Asp541Glu polymorphisms are associated with prostate cancer risk and could be low-penetrance prostate cancer susceptibility biomarkers.

Functional Analysis of a Wilt Fungus Inducible <i>PR</i>10-1 Gene from Cotton

Early stage expression of PR10 combined with phytoalexins contributed to Verticillium wilt resistance in cotton. In order to analysis the activities of PR10 proteins during pathogens’ infection, we cloned a Verticillium-induced PR10 (GbPR10-1) gene from cotton (Gossypium barbadense) and compared its expression patterns and domains with other PR10 proteins. Bioinformatics indicated that GbPR10-1 showed the lowest similarity with other 12 different PR10 genes in cotton (Upland and sea-island cotton). Expression profiles showed that GbPR10-1 gene instantly up-regulated after infection by V. dahliae in the sea-island cotton plants. GbPR10-1 was also induced by environmental stimulus including heat, submergence and salt, and ethylene but not by ABA and salicylic acid. The GbPR10-1 protein expressed in E. coli BL21 demonstrated that it had a low ribonuclease-like activity in vitro, and could inhibit V. dahliae hyphae growth but not its spores. Comparison analysis of GbPR10-1 (from resistant species) and GhPR10-1 (from susceptible species) responding to V. dahliae infection, only GbPR10-1 gene was strongly induced in the sea-island cotton plants (incompatible response), indicating that PR10-1 genes was linked to resistance signal. In summary, the earlier activation of GbPR10-1 gene, as the index of resistance response, would be aid to block

Biological implications of 2-chlorocyclohexa-2,5-diene-1,4-dione toward ribonuclease A

2-Chlorocyclohexa-2,5-diene-1,4-dione (CBQ) or 2-chloro1,4-benzquinone is one of the common metabolites of polycyclic aromatic hydrocarbons generated through industrial processes. This report describes the biological effects of CBQ toward ribonuclease A (RNase). We also investigated the inhibition of RNase modifications and the reactivity of CBQ toward selected amino acids. The study was carried out by incubating RNase or amino acids with CBQ in a concentration- and a time-dependent manner at 37&degC and pH 7.0. SDS-PAGE results showed oligomerization as well as polymeric aggregation of RNase when incubated with CBQ as early as in 10 min. CBQ-induced RNase modifications were inhibited in the presence of NADH or ascorbic acid. CBQ reactivity toward selected amino acids was also evaluated by determining the second-order rate constants for the reactions of CBQ with selected amino acids. It was found that the reactivity toward CBQ decreased in the order of lysine > threonine > serine >> aspartate > cysteine.

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i>against Phytopathogenic Rna-Viruses

Background: Viruses can cause different diseases in plants. To prevent viral infections, plants are treated with chemical compounds and antiviral agents. Chemical antiviral agents usually have narrow specificity, which limits their wide application. Alternative antiviral strategy is associated with the use of microbial enzymes, which are less toxic and are readily decomposed without accumulation of harmful substances. The aim of this work is to study the effect of Bacillus pumilus ribonuclease on various phytopathogenic viruses with specific focus on the ability of enzyme to eliminate them from plant explants in vitro. Materials and methods: Extracellular ribonuclease of B. pumilus is tested as an antiviral agent. To study the antiviral effect of RNase, depending on concentration and the time of application several plant-virus model systems are used. Virus detection is conducted by serological testing and RT-PCR. Results: Bacillus pumilus ribonuclease possesses antiviral activity against plant Rna-viruses RCMV (red clover mottle virus), PVX (Potato Virus X) and AMV (Alfalfa Mosaic Virus). The maximum inhibitory effect against actively replicating viruses is observed when plants are treated with the enzyme in the concentration of 100 ug/ml prior to infection. In case of local necrosis ribonuclease in the concentration of 1 ug/ml completely inhibits the development of RCMV virus on bean plants. The enzyme is able to penetrate plants and inhibit the development of viral infection, inhibiting effect for untreated surfaces decreased on average for 20%. It is also found that B. pumilus ribonuclease protects apical explants of sprouts of potato tubers from PVM and PVS viruses. Conclusion: B. pumilus ribonuclease possesses antiviral activity against plant Rna-viruses and produces viruses-free plants in the apical meristem culture.

水+葡萄糖+电解质三元系统在298.15K下的热力学研究

本文通过电动势法研究了不同浓度的几种电解质从纯水到不同含量的葡萄糖混合溶剂的标准吉布斯自由能的变化，讨论了电解质在混合溶剂中溶质──溶质、溶质──溶剂之间的相互作用．本文发表在国际期刊《ＴｈｅｒｍｏｃｈｉｍｉｃａＡｃｔａ》２２４（１９９３）２６１—２６９中，下面是该文的摘译：

A novel loss-of-function variant in PNLDC1 inducing oligo-astheno-teratozoospermia and male infertility

Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.

Regulation of RNase E during the UV stress response in the cyanobacterium Synechocystis sp.PCC 6803

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression.A key endoribonuclease in Gram-negative bacteria is RNase E.To ensure an appropriate supply of RNase E,some bacteria,such as Escherichia coli,feedback-regulate RNase E expression via the rne 5′-untranslated region(5′UTR)in cis.However,the mechanisms involved in the control of RNase E in other bacteria largely remain unknown.Cyanobacteria rely on solar light as an energy source for photosynthesis,despite the inherent ultraviolet(UV)irradiation.In this study,we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp.PCC ^(6)803 after a brief exposure to UV.Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level.Moreover,the enzymatic activity of RNase E rapidly increased as well,although the protein stability decreased.This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5′UTR and suppression of premature transcriptional termination,but not by an increased transcription rate.Mapping of RNA 3′ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5′UTR,indicating autoregulation.These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.

“Life is short,and art is long”:RNA degradation in cyanobacteria and model bacteria

RNA turnover plays critical roles in the regulation of gene expression and allows cells to respond rapidly to environmental changes.In bacteria,the mechanisms of RNA turnover have been extensively studied in the models Escherichia coli and Bacillus subtilis,but not much is known in other bacteria.Cyanobacteria are a diverse group of photosynthetic organisms that have great potential for the sustainable production of valuable products using CO_(2)and solar energy.A better understanding of the regulation of RNA decay is important for both basic and applied studies of cyanobacteria.Genomic analysis shows that cyanobacteria have more than 10 ribonucleases and related proteins in common with E.coli and B.subtilis,and only a limited number of them have been experimentally investigated.In this review,we summarize the current knowledge about these RNAturnover-related proteins in cyanobacteria.Although many of them are biochemically similar to their counterparts in E.coli and B.subtilis,they appear to have distinct cellular functions,suggesting a different mechanism of RNA turnover regulation in cyanobacteria.The identification of new players involved in the regulation of RNA turnover and the elucidation of their biological functions are among the future challenges in this field.

Parental RNA is Significantly Degraded During Arabidopsis Seed Germination

Germination is the first and maybe the foremost growth stage in the life cycle of a plant. Herein, we report that initiation of germination in the Arabidopsis Columbia ecotype was accompanied by a sharp decrease in the amount of extractable total RNA. At the beginning of our germination experiment, we were usually able to obtain 35-40 IJg total RNA from 100 mg dry seeds. However, after 3 d of cold stratification, we could only obtain less than 5 μg total RNA from the same amount of starting material. Young seedlings contained approximately 100 μg total RNA per 100 mg fresh tissue. Further studies showed that inhibition of de novo RNA synthesis by actinomycin D prevented the degradation of parental RNA and, in the meantime, significantly delayed the germination process. Several ribonuclease-like genes that were highly expressed in dry seeds, and especially during the cold stratification period, were discovered. We propose that these enzymes are involved in the regulation of parental RNA degradation. These results indicate that parental RNA metabolism may be an important process for Arabidopsis seed germination.

Regnase-1, a rapid response ribonuclease regulating nflammation and stress responses

RNA-binding proteins （RBPs） are central players in post-transcriptional regulation and immune homeostasis. The ribonuclease and RBP Regnase-1 exerts critical roles in both immune cells and non-immune cells. Its expression is rapidly induced under diverse conditions including microbial infections, treatment with inflammatory cytokines and chemical or mechanical stimulation. Regnase-1 activation is transient and is subject to negative feedback mechanisms including proteasome-mediated degradation or mucosa-associated lymphoid tissue 1 （MALT1） mediated cleavage. The major function of Regnase-1 is promoting mRNA decay via its ribonuclease activity by specifically targeting a subset of genes in different cell types. In monocytes, Regnase-1 downregulates IL-6 and IL-12B mRNAs, thus mitigating inflammation, whereas in T cells, it restricts T-cell activation by targeting c-Rel, Ox40 and 11-2 transcripts. In cancer cells, Regnase-1 promotes apoptosis by inhibiting anti-apoptotic genes including Bcl2L1, Bcl2A1, RelB and Bcl3. Together with up-frameshift protein-1 （UPF1）, Regnase-1 specifically cleaves mRNAs that are active during translation by recognizing a stem-loop （SL） structure within the 3＇UTRs of these genes in endoplasmic reticulum-bound ribosomes. Through this mechanism, Regnase-1 rapidly shapes mRNA profiles and associated protein expression, restricts inflammation and maintains immune homeostasis. Dysregulation of Regnase-1 has been described in a multitude of pathological states including autoimmune diseases, cancer and cardiovascular diseases. Here, we provide a comprehensive update on the function, regulation and molecular mechanisms of Regnase-1, and we propose that Regnase-1 may function as a master rapid response gene for cellular adaption triggered by microenvironmental changes.

Fg12 ribonuclease secretion contributes to Fusarium graminearum virulence and induces plant cell death

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean;its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light-dependent manner. Fg12 possessed ribonuclease(RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death-promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.

Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2

Mammalian mitochondrial genome encodes a small set of tRNAs, rRNAs, and mRNAs. The RNA synthesis process has been well characterized. How the RNAs are degraded, however, is poorly understood. It was long assumed that the degradation happens in the matrix where transcription and translation machineries reside. Here we show that contrary to the assumption, mammalian mitochondrial RNA degradation occurs in the mitochondrial intermembrane space （IMS） and the IMS- localized RNASET2 is the enzyme that degrades the RNAs. This provides a new paradigm for understanding mitochondrial RNA metabolism and transport.

Adaptive evolutionary expansion of the ribonuclease 6 in Rodentia

Ribonuclease 6(RNase6 or RNase K6)is a protein that belongs to a superfamily thought to be the sole verte­brate-specific enzyme known for a wide range of physiological functions,including digestion,cytotoxicity,an­giogenesis,male reproduction and host defense.In our study,51 functional genes and 11 pseudogenes were identified from 27 Rodentia species.Intriguingly,in the 3 main lineages of rodents there were multiple RNas­e6s identified in all species of Ctenohystrica,whereas only a single RNase6 was observed in other Rodentia spe­cies examined except for 2 species in the mouse-related clade.The evolutionary scenario of“birth(gene dupli­cation)and death(gene deactivation)”and gene sorting have been demonstrated in Ctenohystrica.In addition,bursts of positive selection,diversification of isoelectric point and positive net charge have been identified in Ctenohystrica,especially at two key sites that are involved in antimicrobial function.Site Trp30 has undergone positive selection and Ile45 has changed into other residues in Group B and Group C of the Ctenohystrica.Our results demonstrated a complex and intriguing evolutionary pattern of rodent RNase6,and indicated that func­tional modification may have occurred,which establishes an important theoretical foundation for future func­tional assays in rodent RNase6.

Fourier Transformed Infrared Spectra of the Unfolding of Ribonuclease A in Guanidinium Chloride

The unfolding of proteins during denaturation by guanidine or urea has been extensively studied. However, the methods hitherto employed usually provide only a limited amount of information on gross changes of such molecular properties as shape, size, or the exposure of buried aromatic residues. CD studies are hampered by high absorption of the denaturants commonly employed in the far ultraviolet region. Recent development in Fourier